The present invention relates to novel synthetic binding pairs, to methods of preparing same and to uses thereof in various applications such as, but not limited to, isolation and purification of biological molecules via affinity chromatography, immunohistochemical staining, introducing multiple labels into tissues, localizing hormone binding sites, flow cytometry, in situ localization and hybridization, radio-, enzyme-, and fluorescent immunoassays, neuronal tracing, genetic mapping, hybridoma screening, purification of cell surface antigens, coupling of antibodies and antigens to solid supports, examination of membrane vesicle orientation, and drug delivery.
High-affinity and specificity pairs are of great importance in both research and industrial endeavors in fields such as chemistry and, in particular, in the biological and medical sciences. Affinity chromatography alone is a valuable tool for separating and purifying biological materials from solution. Affinity chromatography technique typically involves an affinity pair, of which one component (oftentimes referred to as the ligand) is immobilized by attaching it to an insoluble support. The other component, when passed through a column within a mixture of components in solution, is selectively absorbed to the attached component by forming a complex therewith and is thus isolated from the solution. The second component may subsequently be eluted from the solid support by a number of procedures resulting in the dissociation of the affinity pair.
This technique is widely used to isolate biomolecules such as peptides, proteins, enzymes, inhibitors, antibodies, antigens, hormones, carbohydrates and many more, based on specific interactions formed between affinity pairs of such biomolecules. Known examples are the high-affinity and specificity interactions of antibody-hapten pairs, and in particular, of the avidin-biotin (Av-B) pair (Wilchek M, Methods Enzymol., entire Volume 184, 1990, incorporated by reference as if fully set forth herein).
The Av-B high affinity complexation and the consequent stability of its non-covalent interactions (KD of 1015 M−1) has become the basis of a broad variety of bioanalytical applications and a common tool in almost any molecular biology laboratory. The main applications where Av-B complexes have been used extensively include, for example, isolation via affinity chromatography, localization via cytochemistry, cell cytometry, in situ hybridization and blotting technologies, diagnostics via immunoassay, histochemistry and histopathology, and gene probes. In addition, the Av-B complexes have been also applied in the hybridoma technology, in the design of bioaffinity sensors, in affinity targeting, drug delivery, cross linking, immobilization, fusogenic studies, screening of combinatorial libraries, in vivo tissue imaging and many more.
Biotin is a relatively small molecule, a member of the Vitamin B family (formerly known as Vitamin H), whereby avidin is a ubiquitous 66 kD tetrameric protein found in egg whites. A key principle of the Av-B technology is that both avidin and biotin can be chemically linked to a variety of either small or large molecules without disrupting the binding constant therebetween. For example, many reporter groups have been covalently attached to avidin, including fluorescent groups, electron-dense markers, enzymes, various binding protein, and various solid supports including magnetic beads. Likewise, since the carboxyl group of biotin is not essential for binding, it has been used either directly or through a spacer fragment, to synthesize many compounds including proteins, DNA and RNA molecules, with a covalently attached biotin moiety.
By covalently attaching a biotin molecule, a reaction known as biotinylation, one can “tag” an otherwise untraceable molecule or a biochemical entity and turn it into a probe that can be recognized by a labeled detection reagent or an affinity-capture matrix. Once tagged with biotin, a molecule of interest, such as a peptide, a protein, an antibody, a drug, a polynucleotide, a polysaccharide or another receptor ligands, can be used to probe complex systems and mixtures, cells and tissues, as well as protein and nucleic acid blots and arrays. This tagged molecule can then be detected with the appropriate avidin conjugate that has been labeled with a chromophore/fluorophore, enzyme or other solid and/or magnetic matrices and particles. Biotinylated molecules can also be captured with various forms of immobilized avidin or streptavidin, and other modified forms of avidin.
Although binding of biotin to native avidin or streptavidin is essentially irreversible, appropriately modified avidins can bind biotinylated probes reversibly, making them valuable reagents for isolating and purifying biotinylated molecules from complexed mixtures (Morag E, Bayer E A, Wilchek M. Biochem J 316, 193-199 (1996)).
Many strategies are available for applying the Av-B technology in a given experimental system. Representative examples of these strategies are presented in FIG. 1. Thus, in one exemplary strategy (FIG. 1, Strategy A) avidin is attached to a probe, either directly, by covalent bonding, or indirectly, via interaction with a biotinylated probe, and the target molecule is directly bound to biotin. The biotinylated target molecule forms a complex with the avidin probe and is thus analyzed. In another exemplary strategy (FIG. 1, Strategy B) a target molecule is attached to a ligand which is covalently bound to either biotin or avidin in order to generate a noncovalent linkage to an avidin-probe conjugate, as described hereinabove, or a biotin probe conjugate, respectively. In yet another exemplary strategy (FIG. 1, Strategy C) the same principles as in B are utilized, but the target molecule is further attached to a binder that is specific to the first ligand, thus generating a longer chain of interactions.
However, although technologies utilizing the Av-B affinity pair has been extensively used over the past two decades, they suffer several disadvantages, the following lists a few.
The Av-B binding couple exhibits a disadvantageous high molecular weight, with 58-76 kD for Avidin and 244 D for Biotin. Such a high molecular weight may lead to loss of resolution in separation techniques, analytical gels and other applications where the studied and compared components are small relative to avidin.
The fact that avidin and streptavidin are large biological macromolecules, characterized by a complexed yet inflexible structure, renders these biomolecules susceptible to interactions with many other small molecules and biomolecules. For example, due to the molecular orientation of the binding sites, less than four molecules of biotin actually bind to one avidin molecule. The few binding sites on avidin are also the sites where chemical modification takes place, limiting the capacity for labels and affecting immobilization properties. Avidin may also bind many other biomolecules non-specifically. This is especially significant in the case of preparation of oligonucleotide microarrays in which non-biotin modified oligonucleotides bind non-specifically to avidin, leading to spurious results.
Furthermore, the fact that proteins are sensitive to chemical and physical conditions renders the use of avidin or streptavidin limited to technologies that involve mild conditions and limits the use of the Av-B affinity pair to aqueous systems at close to physiological temperature.
In addition, Av-B systems suffer from lack of transparency in the UV region and background fluorescence, and therefore cannot be used in experiments where detection is depending on various light measurements.
The conjugation chemistry of avidin is rather limited to reactions that are compatible with polypeptides, and thus limits the chemistry by which avidin can be attached to various molecules and materials.
The resulting affinity of the Av-B system has an inflexible binding constant of KD=10−15 M, which limits their use to applications that require strong binding without the possibility for fine-tuning.
The high binding affinity decreases rapidly (100-1000 fold) when a biomolecule is coupled to biotin.
Finally, the one-step binding of the Av-B couple is practically irreversible, unless large quantities of free biotin are applied. The irreversible nature of the binding limits the ways by which the dissociation of the conjugate can be achieved.
Cucurbiturils are macrocyclic cavitand compounds that are typically formed by reacting a number of glycoluril units and formaldehyde units under acidic conditions. For example, Cucurbit[6]uril, also known as CB[6] (FIG. 2, Compound 1), is typically prepared by reacting six glycoluril molecules, (FIG. 2, Compound 2) and twelve formaldehyde units, in the presence of a concentrated acid, as is illustrated in FIG. 2.
Cucurbiturils (CBs) in general are known since 1905 (Behrend et al., Liebigs Ann. Chem. 1905, 339, 1), and were first characterized by Mock and co-workers in 1981 (Mocket et al., J. Am. Chem. Soc. 1981, 103, 7367). Several substituted cucurbiturils and homologues, collectively referred to as CB[n] whereby n represents the number of glycoluril units in the CB and typically ranges from 5 to 8, have also been prepared and characterized (Kim, et al., J. Am. Chem. Soc. 2000, 122, 540).
Cucurbiturils, either substituted or unsubstituted, are typically characterized by a hydrophobic cavity that is accessible through two identical, polar, carbonyl-fringed portals. This feature, when coupled with the high yield synthesis of, for example, CB[6] (82%), suggested that the formation of CB[6] is governed by a thermodynamic preference for CB[6] (Buschmann et al., German Patent DE 196 03 377 A1, 1997). Other studies further indicated that the ring order and by-product population proportions in cucurbiturils syntheses are determined by the glycoluril and aldehyde building-blocks substitution (see, U.S. Pat. No. 6,639,069, and Chakraborty et al., J. Am. Chem. Soc. 2002, 124, pp. 8297-8306).
Although cucurbiturils are easily prepared via an acid-catalyzed condensation of the appropriate glycolurils with formaldehyde, these macropolycyclic compounds are typically obtained in the form of complex mixtures that further contain many cyclic and acyclic oligomers and polymers, including insoluble polymers.
The presently known methods of isolating CB[n]s from their reaction mixtures are based mainly on differential solubility in various solvents and on fractional crystallization, methods which suffer from low efficiency in terms of cost and yield. These methods, however, are oftentimes not suitable for isolation and purification of substituted CB[n]s. The use of alternative purification or isolation methods such as, for example, column chromatography is, in most cases, inefficient and difficult to practice due to the high polarity and limited solubility of these compounds.
Thus, the isolation of pure CB[n]s has become the major impediment to their availability, particularly when large-scale synthesis is required.
U.S. Pat. No. 6,365,734 describes the preparation and separation of various CB[n] homologues and derivatives. These methods involve manipulation of reaction conditions, e.g. acidity and temperature, which cause a shift in the proportions between various major and minor products, yet these manipulations do not provide an efficient method for obtaining, in substantial amounts, minor, thermodynamically disadvantageous CB[n]s, which are typically formed in traces amounts.
The rigid structure and the combination of a hydrophobic cavity with polar portals allow the cucurbiturils to act as cavitands hosting various molecules and cations, and thus render the CB[n]s attractive synthetic receptors and useful building blocks of various supramolecular structures.
Due to the intricate recognition characteristics of CB[n], many studies were aimed at synthesizing homologues and derivatives of CB[n] with varying ring order and substituents. Nevertheless, the domination of one major product, and the practical difficulty in separating the more desired yet minor products, presented major restrictions on the path to obtaining rare CB[n]s.
As mentioned hereinabove, CB[n]s are characterized by two “oculi”, having a 400 pm diameter in the case of CB[6]. These openings allow the entrance of small molecules into the cavity and thus enable an affinity binding of these molecules to the cavitand. Although simple aliphatic compounds can thus be bound, the most strong and efficient affinity binding in the cavity of CB[n]s has been observed with alkylammonium ions (Mock and Shih, J. Org. Chem. 1983, 48, p. 3618).
The exceptional binding affinity between alkylammonium ions and CB[n]s has been attributed to the ion-dipole interaction between the ammonium moiety and the oculi carbonyls, and to the hydrophobic interactions formed when the alkyl moiety displaces solvent molecules from within the cavity (Mock and Shih, J. Org. Chem. 1986, 51, p. 4440).
The combination of complexation and recognition interactions lends itself to a range of strong and highly specific entrapping abilities of n-alkylammonium ions by various cucurbiturils. The symmetric structure of the two “oculi” further offers recognition factors, as evident from the interaction of n-alkyldiammonium ions with CB[n] (Mock, W. L. in Comprehensive Supramolecular Chemistry; Vögtle, F., Ed.; Elsevier Press: New York, 1996; Vol. 2, pp 477-493).
Studies have shown (Mock and Shih, J. Org. Chem. 1986, 51, p. 4440) that the binding strength between alkylammonium ions and cucurbiturils depends on the chain length of the alkyl group of n-alkylammonium and n-alkyldiammonium ions, whereby the optimal chain length was found to be 4, for n-alkylammonium, and 5-6 for n-alkyldiammonium, with the latter possessing ten-fold higher affinity to CB[6] as compared with that of the first.
The binding affinity between alkylammonium ions and CB[n]s was found to be further affected by stearic hindrance and ring size, in cases of substituted and cyclic ammonium ions (Mock and Shih, J. Org. Chem. 1986, 51, p. 4440). Thus, the presence of two or more amine groups in the alkyl chain, was found to affect the binding rate and dynamics, being a domain of distinguished states sensitive to chemical (e.g., pH) and physical (e.g., temperature) conditions, and thus rendering CB[n]-polyamine systems highly suitable for molecular switches and quantum binding.
CB[n]s and protonated polyaminoalkanes form stable host-guest complexes, exhibiting sub-micromolar affinity dissociation constants (KD) in the range of e.g., 10−5-10−7 M (Mock et al, J. Org. Chem. 1986, 51, 4440) for protonated diaminoalkanes, such as 1,4-diaminobutane, 1,5-diaminopentane and 1,6-diaminohexane. This property has been extensively used by Kim [1] and others [2] to construct many supramolecular assemblies, including catenanes, rotaxanes, and pseudopolyrotaxanes.
Nevertheless, although the high affinity between CB[n]s and polyamines has been studied, the use of CB[n]s-polyamines affinity pairs both in basic research and in biotechnology and medicine applications such as, for example, tagging and labeling, purification, cytometry, drug delivery, administration and other applications, has never been suggested nor practiced hitherto.
While conceiving the present invention, it was envisioned that the high affinity between CB[n]s and polyamines, the versatile and controllable characteristics of CB[n]s and polyamines and the effect of these characteristics on the affinity could be beneficially used in a myriad application, while circumventing the limitations associated with the presently used Av-B technology.
There is thus a widely recognized need for, and it would be highly advantageous to have a practical, fast, general and cost effective method for separation and purification of CB[n] homologues or derivatives, which would enable to obtain CB[n]s and in particular rare and thermodynamically disadvantageous forms of CB[n]s, in substantial yields, while circumventing the above limitations, and which would enable to efficiently use such CB[n]s to form high-affinity pairs of CB[n] entities and protonated polyaminoalkanes, devoid of the limitations associated with the presently known affinity pairs.